The antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) have been associated for some time with cardiovascular as well as other tissue toxicities that appear to be directed at mitochondrial biogenesis. In particular, studies have demonstrated that mitochondrial DNA is depleted during toxicity, suggesting a block in DNA replication. Since the triphospho-forms of these nucleoside analogs have been shown to varying degrees to directly inhibit mitochondrial DNA polymerase, this has generally been promoted as the target of this toxicity. However, as discussed in this proposal, we have shown that one of the HAART components, AZT (Zidovudine) is converted slowly only to AZT-MP and no further, in both isolated heart mitochondria and in the isolated perfused rat heart under conditions in which labeled thymidine is readily converted to labeled TTP in both models. Thus it seems unlikely that the toxicity of AZT in the heart is mediated by AZT-TP inhibition of polymerase. Rather, we have shown that the presence of AZT, perhaps via AZT-MP, is a potent inhibitor of thymidine phosphorylation in both isolated heart mitochondria and in the isolated perfused heart. These data suggest an alternative hypothesis that the toxicity of AZT and perhaps other analogs, is caused by their interference with the metabolism of endogenous deoxynucleosides disrupting the substrate supply of dNTPs for mitochondrial DNA replication. This grant has three major goals. The first goal is to document and quantitate the uptake and conversion of each of the endogenous deoxynucleosides, and each of the nucleoside analogs used in HAART therapy, to their tri-phospho-forms in isolated heart mitochondria and in the isolated perfused rat heart. From these data we can determine the relative role of the mitochondria in the conversion process and we can estimate the levels of the various analog intermediates and determine if there is sufficient synthesis of the triphosphate to inhibit the mitochondrial polymerase. The second goal of this proposal is to evaluate the effect of these same nucleoside analogs on the uptake and phosphorylation of the endogenous deoxynucleosides in isolated heart mitochondria and in the isolated perfused heart. The third goal is to evaluate the effect of combination drug therapy on analog and endogenous deoxynucleoside phosphorylation. From this work we hope to identify both the potential toxic form of the nucleoside analog as well as its potential enzymatic target.